1. Field of the Invention
The invention relates to nucleic acid molecules, plasmids (e.g., bi-cistronic plasmids), host cells and methods used for the expression of formate dehydrogenase (FDH) and modified phenylalanine dehydrogenase (PDHmod).
2. Background of the Invention
Dipeptidyl peptidase IV is a membrane bound non-classical serine aminopeptidase which is located in a variety of tissues including, but not limited to, intestine, liver, lung, and kidney. This enzyme is also located on circulating T-lymphocytes wherein it is referred to as CD-26. Dipeptidyl peptidase IV is responsible for the metabolic cleavage of the endogenous peptides GLP-1(7-36) and glucagons in vivo and has demonstrated proteolytic activity against other peptides such as GHRH, NPY, GLP-2 and VIP in vitro.
GLP-1(7-36) is a 29 amino acid peptide derived from post-translational processing of proglucagon in the small intestine. This peptide has multiple actions in vivo. For example, GLP-1(7-36) stimulates insulin secretion and inhibits glucagon secretion. This peptide also promotes satiety and slows gastric emptying. Exogenous administration of GLP-1(7-36) via continuous infusion has been shown to be efficacious in diabetic patients. However, the exogenous peptide is degraded too rapidly for continual therapeutic use.
Inhibitors of dipeptidyl peptidase IV have been developed to potentiate endogenous levels of GLP-1(7-36). U.S. Pat. No. 6,395,767 discloses cyclopropyl-fused pyrrolidine-based inhibitors of dipeptidyl peptidase IV. Methods for chemically synthesizing these inhibitors are disclosed in U.S. Pat. No. 6,395,767 as well as in the literature. For example, see Sagnard et al. Tetr. Lett. 1995 36:3148-3152; Tverezovsky et al. Tetrahedron 1997 53:14773-14792; and Hanessian et al. Bioorg. Med. Chem. Lett. 1998 8:2123-2128. An example of an inhibitor disclosed in U.S. Pat. No. 6,395,767 is the free base, (1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-tricyclo[3.3.1.1.3,7]dec-1-yl)-1-oxoethyl]-2-azabicyclo-[3.1.0]hexane-3-carbonitrile.
Methods for preparing intermediates used in the production of this dipeptidyl peptidase IV inhibitor are disclosed in EP 0 808 824 A2. Also see, Imashiro and Kuroda Tetrahedron Letters 2001 42:1313-1315, Reetz et al. Chem. Int. Ed. Engl. 1979 18:72, Reetz and Heimbach Chem. Ber. 1983 116:3702-3707, Reetz et al. Chem. Ber. 1983 116:3708-3724.
Recombinant expression of the enzymes formate dehydrogenase (FDH) (e.g., from Pichia pastoris (ATCC 20864)) and phenylalanine dehydrogenase (PDH) (e.g., from Thermoactinomyces intermedius (ATCC 33205)) can be used in the biotransformation of 3-hydroxy-α-oxotricyclo-[3.3.1.13,7] decane-1-acetic acid to (αS)-α-amino-3-hydroxytricyclo[3.3.1.13,7] decane-1-acetic acid which is then chemically converted to (αS)-α-[[(1,1 dimethylethoxy)carbonyl]amino]-3-hydroxytricyclo[3.3.1.13,7] decane-1-acetic acid, a starting material in the synthesis of the DPPIV inhibitor, saxagliptin. Previous attempts to express FDH and PDH using a single plasmid containing two identical tandem promoters (one promoter for each enzyme) to drive expression have had limited success. In particular, prior attempts appear to have resulted in bacterial cultures harboring two different populations of bacterial cell: one containing an intact plasmid, and another wherein the portion of the plasmid containing the FDH gene appears to have been processed (truncated). The processing of the plasmid appears to have increased with each cell generation.
Thus, while biotransformation methods have been identified as a method for generating complex chemical precursors, the biosynthetic processes discussed above suffer decreased product yield because of genetic instability. Thus, there is a need for alternative recombinant sequences and methods for generating synthetic precursor molecules in the eventual production of chemical compounds, such as saxagliptin.